Methods and compositions for the treatment of beta-thalassemia

ABSTRACT

Methods and compositions for the treatment of β-thalassemia are provided. Methods and compositions restore or increase erythrocyte maturation in individuals afflicted with β-TM by preventing proteolysis of GATA-1 protein. Screening methods for identifying agents which bind heat shock protein 70 (HSP-70) and inhibit HSP-70 α-globin binding, but which allow GATA-1 protein-HSP-1 binding in a manner that prevents GATA-1 proteolysis.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to the treatment of β-thalassemia(β-TM). In particular, the invention provides methods and compositionsfor restoring or increasing erythrocyte maturation in individualsafflicted with β-TM by preventing proteolysis of GATA-1 protein.

BACKGROUND OF THE INVENTION

Adult mammalian Hg is a multimeric protein that includes two α and two βglobin chains which together form the (α/β)₂ tetrameric hemoglobin (Hb)molecule. Beta-thalassemias are a group of inherited blood disorderscaused by a quantitative defect in the synthesis of the β chains ofhemoglobin. In individuals with this disorder, the synthesis of β-globinchains is reduced or absent. Three main forms of the disease have beendescribed: β-thalassemia major (β-TM or β⁰-TM) in which no β chain isproduced, and β-thalassemia intermedia and β-thalassemia minor, in whichβ chain is produced but in lower than normal amounts. These conditionscause variable phenotypes ranging from severe anemia to clinicallyasymptomatic individuals. Individuals with β-TM usually present withinthe first two years of life with severe anemia, poor growth, andskeletal abnormalities during infancy. Affected children will requireregular lifelong blood transfusions. β-thalassemia intermedia is lesssevere than β-thalassemia major and may require episodic bloodtransfusions. Transfusion-dependent patients will develop iron overloadand require chelation therapy to remove the excess iron.

It is known that defective β-globin chain synthesis leads to theaccumulation of free α-globin chains that form toxic aggregates^(1,2).However, despite extensive knowledge on the molecular defects causingβ-TM, little is known about the mechanisms responsible for ineffectiveerythropoiesis (IE) in β-TM patients. In such individuals,erythropoiesis does not result in the production of mature erythrocytesbut instead is characterized by accelerated erythroid differentiation,maturation arrest and apoptosis at the polychromatophilic stage³⁻⁵. Thislack of understanding of the mechanism has prevented the development ofeffective strategies for treating the disease.

Humans are capable of producing three types of Hb chains: α, β and γ.The main oxygen transport protein in the human fetus during the lastseven months of development in the uterus and in the newborn untilroughly 6 months of age is (α/γ)₂Hb. As the nomenclature indicates, thistype of Hb tetramer contains two α globin subunits and two γ globinsubunits. After about 6 months of age, humans shift from production of(α/γ)₂ Hb toward production of (α/β), Hb, and in non-β-TM adult humans,(α/γ)₂ Hb represents only about 1% or less of hemoglobin. However, theamount of (α/γ)₂ Hb is increased in individuals with β-TM. Functionally,fetal hemoglobin differs from adult hemoglobin in that it is able tobind oxygen with greater affinity than the adult form, giving thedeveloping fetus better access to oxygen from the mother's bloodstream.Thus, the production of (α/γ)₂ by β-TM cells could, in theory, be a boonfor those suffering from thalassemias. However, since β-TM erythrocytesgenerally fail to mature, the presence of the alternate form of Hb isnot especially useful to patients with this disease.

SUMMARY OF THE INVENTION

Individuals afflicted with the genetic disease β-TM do not producehemoglobin β chains. Thus, mature (α/β)₂ Hb cannot be formed and the αglobin chains that are produced accumulate in the cytoplasm of immatureerythrocytes (erythroblasts). Up until the present invention, therelationship between α chain accumulation and the etiology of β-TM wasunknown. The lack of knowledge greatly hampered the development ofeffective treatment regimes for β-TM patients.

The present inventors have elucidated the consequences of α chaincytoplasmic accumulation and the cascade of failed reactions that resulttherefrom which ultimately cause β-TM symptoms such as anemia. Thediscovery is based on the further clarification of the roles of thechaperone protein Hsp70 and the erythrocyte maturation protein GATA-1.The inventors have discovered that Hsp70 has important functions in boththe cytoplasm and the nucleus of erythroblasts. A primary function ofHsp70 in the nucleus is binding to the GATA-1 protein and preventing itscleavage and proteolytic degradation (by the protease caspase-1). GATA-1thus prevents inactivation of GATA-1 and preserves its function as a keyfactor in erythrocyte maturation. A secondary function of Hsp70 isbinding to α globin in the cytoplasm and ensuring that the proteinchains are properly folded and can form tetrameric (α/β)₂ Hb.Ordinarily, there is sufficient Hsp70 available in the cell to carry outboth of these functions. However, in β-TM cells, the Hsp70 ismonopolized by the excess free α chains which accumulate in thecytoplasm. Thus, a disproportionate amount of the Hsp70 is sequesteredin the cytoplasm, and there is not sufficient Hsp70 available forbinding and protecting GATA-1 in the nucleus. Unprotected GATA-1 isproteolytically cleaved and inactivated, and proper erythrocytematuration does not occur. Rather, the absence of active GATA-1 resultsin maturation arrest and apoptosis of immature erythrocytes at thepolychromatophilic stage. This sequence of events is thus initiallytriggered by a lack of hemoglobin β chains and ultimately results in low(or no) erythrocyte production, causing anemia.

The present invention provides methods and pharmaceutical compositionsdesigned to intervene in this defective process and to promote orrestore erythrocyte maturation in individuals suffering from β-TM. It isnoted that because β globin is not formed in β-TM erythrocytes, the typeof erythrocytes that are produced in individuals treated with themethods and compositions described herein contain (α/γ)₂ Hb, and theinvention provides methods and compositions for increasing theproduction of (α/γ)₂ Hb erythrocytes in β-TM cells and β-TM individuals.The methods involve maintaining the activity of GATA-1 by preventing itsproteolysis, e.g. by preventing sequestration of Hsp70 in the cytoplasm.

Accordingly, it is an object of this invention to provide methods ofrestoring or increasing erythrocyte maturation in a subject sufferingfrom β-thalassemia major (β-TM) by preventing proteolytic inactivationof GATA-1. In some embodiments, preventing is achieved by administeringto the subject a compound that inhibits binding of α globin to Hsp70. Inan exemplary aspect, the compound that inhibits binding of α globin toHsp70 is a small molecule that binds to the α chain binding pocket ofHsp70. The invention also includes screening methods to identify suchagents.

Other features and advantages of the present invention will be set forthin the description of invention that follows, and in part will beapparent from the description or may be learned by practice of theinvention. The invention will be realized and attained by thecompositions and methods particularly pointed out in the writtendescription and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Hsp70 and GATA-1 expression in fresh bone marrow ofβ-thalassemia major patients (β-TM). (A) Representative confocalmicroscopy analysis of α-globin, Hsp70 and GATA-1 expression in freshbone marrow (BM) from 3 adult β-TM patients and 3 healthy donors. Scalebar, 5 μm. Gray arrows indicate Hsp70 cytoplasmic sequestration intomature β-TM cell, and the white arrow shows immature cell. (B) Hsp70cytoplasmic/nuclear mean fluorescence intensity (MFI) ratio (top) andGATA-1 nuclear MFI (bottom) of β-TM and controls. Bars indicate themedian (IQR). p values were calculated using the Mann-Whitney U-test.**p<0.01.

FIGS. 2A-D. The characteristics of ineffective erythropoiesis in β-TMand the kinetics of Hsp70 and GATA-1 expression during in vitroerythroid differentiation. CD36⁺ cells derived from CD34⁺ adult β-TMperipheral blood cells (n=16 independent experiments, 7 patients) orhealthy peripheral blood cells (controls, n=8) were cultured with atwo-phase amplification liquid culture system, as described in Methodssection. Day 0 of differentiation is the start of CD36⁺ cell culture andrepresents the differentiation phase of the culture system. (A)May-Grünwald-Giemsa staining at day 8. Left panel. A representativemorphological analysis (×40) of erythroid differentiation is shown.Solid arrows indicate reticulocytes, dashed arrows represent acidophiliccells, and dotted arrows represent polychromatophilics cells. Upperright panel: graph represents proportion of each erythroid stage in β-TMor control-derived cells. Proerythtroblast (ProE)+basophilic (baso),polychromatophilics (Poly) cells and very mature cells-acidophilic cells(Acido)+reticulocytes (Retic) are expressed as a percentage of the totalerythroid cells. Bars indicate the mean ±SEM for 14 independentexperiments. p values were calculated using t-tests. **p<0.01;***p<0.001. Lower right panel: graph represents the terminal maturationindex of β-TM and control-derived cells, as calculated by (acidophiliccells+reticulocytes per slide)×100/polychromatophilic cells per slide.The box and whiskers indicate the median extremes. **p<0.01. (B) Upper:apoptosis curves assessed daily by flow cytometry analysis of Annexin-V(AV) staining (mean percentage±SEM of AV positive, Propidium Iodide (PI)negative cells). Lower: differentiation curves assessed by benzidinestaining. The results are the means of 17 independent experiments (meanpercentage±SEM of positive cells). (C) Left: representative confocalmicroscopy analysis of Hsp70, GATA-1 and α-globin at day 8.The mergedpanel shows Hsp70 and α-globin co-localisation in the cytoplasm. Bargraphs at the right show: upper, Hsp70 cytoplasmic/nuclear MFI ratioand; lower, nuclear GATA-1 MFI of β-TM and control-derived cells. Barsindicate the median (IQR) of 15 independent experiments. p values 15were calculated using the Mann-Whitney U-test. ***p<0.001. (D) Kineticsof Hsp70 and GATA-1 expression in vitro. Hsp70 cytoplasmic/nuclear MFIratio and GATA-1 nuclear MFI were analysed at days 3, 5 and 8 ofculture: right, 3 β-TM patients; and left, 3 controls. Bars indicate themedian (IQR). p values were calculated using the Maim-Whitney U-test.*p<0.05, **p<0.01; ***p<0.001.

FIG. 3A-C: Hsp70 and α-globin interaction. (A) DUOLINK® in situproximity ligation assay (PLA). Interactions were analysed at day 8 inCD36⁺ β-TM or control-derived cell cultures by PLA, according to themanufacturer's instructions using Hsp70 (1:200) and α-globin (1:200)antibodies. A FITC-conjugated Annexin-V antibody (1:200) was added atthe end of the assay. The spots indicate close proximity (<40 nm)between cellular bound antibodies. A representative experiment is shown(n=3). Nuclei are stained with DAPI, and Annexin-V staining is alsoshown. Scale bar, 5 μm. (B) Yeast two-hybrid assay detected, in upperpanel, a direct interaction of α- and β-globin chains with Hsp70(diploid yeast cells), but not, in lower panel, with the Hsp70 deletionmutants, Hsp70-Nucleotide Binding Domain (NBD) or Hsp70-SubstrateBinding Domain (SBD). Each section contains diploid yeast cellsresulting from an independent yeast mating experiment with thecorresponding bait protein and prey protein. The empty vectors, pGADT7and pGBKT7, are used as negative controls. SV40 and p53 coding sequencesare used as positive controls. (C) Structural modelling of Hsp70 and theHsp70¹⁻⁷⁰¹/α-globin complex. In the upper panel, from left to right, arethe molecular surface and cartoon representation of the generated modelsof Hsp70, the Hsp70/α-globin complex and their superimposition. Thedifferent Hsp70 domains are labelled and highlighted. The lid, the SBD,the NBD, the hinge region between the NBD and the SBD, and α-globin areshown. The superimposed models are presented in transparent (Hsp70) andcontrast (Hsp70/α-globin complex) modes. The lid and SBD movements areshown by arrows. The haem group and ADP are shown by space-fillingmodels. In the lower panel, from left to right, are the electrostaticpotential of the molecular surfaces of the Hsp70¹⁻⁷⁰¹/α-globin complex,where Hsp70 and α-globin are shown. The α-globin in theHsp70¹⁻⁷⁰¹/α-globin complex is shown for clarity. α-globin is rotated180° respectively to its position in the Hsp70¹⁻⁷⁰¹/α-globin complex,and its image is flipped horizontally to show the charge distribution onthe surface interacting with Hsp70.

FIG. 4A-D. The transduction of a nuclear Hsp70 mutant (Hsp70-S400A) or acaspase resistant GATA-1 mutant (μGATA-1) rescues cell terminalmaturation and cell survival in β-thalassemia major (β-TM). β-TM CD34+cells were transduced with a nuclear-targeted Hsp70 lentiviral mutant(Hsp70-S400A), a retroviral mutant of GATA-1 uncleavable by activatedcaspase-3 (μGATA-1), and their appropriate empty vectors as controls andwere cultured as described in the methods. CD36⁺/GFP⁺ cells were thenpurified, cultured, and differentiated in Epo-containing medium. Alldata presented here were analysed at day 7 of the CD36⁺ culture. (A) Aconfocal microscopy image of Hsp70 and GATA-1 is shown in the leftpanel. The image is representative of three experiments. Graphs in theright panel represent the nuclear mean fluorescence intensity (MFI) ofHsp70 and GATA-1 (±95% CI) in transduced and control cells (top panel:Hsp70-S400A transduced cells; bottom panel: μGATA-1 transducted cells)(20 cells were analysed in three different fields/slide, n=3 patients).Scale bar, 5 μm. P values were calculated using Mann-Whitney U-test.***p<0.001. (B) May-Grünwald-Giemsa (MGG) staining at day 7. Left panel:Graphs represent proportion of each erythroid stage in transduced andcontrol β-TM cells (upper panel, Hsp70-5400A; lower panel, μGATA-1).Proeryhtroblast (ProE)+basophilic (baso), polychromatophilics (Poly)cells and very mature cells-acidophilic cells (Acido)+reticulocytes(Retic) are expressed as a percentage of the total erythroid cells. Barsindicate the mean±SEM for 3 independent experiments. p values werecalculated using t-tests. Right panel: one representative morphologicalanalysis of the erythroid differentiation for each transduction (n=3) isshown (×100). Solid arrows indicate reticulocytes, dashed arrowsindicate acidophilic cells, and dotted arrows indicatepolychromatophilic cells. (C) Apoptosis in the GFP⁺/propidiumiodide-cell population was assessed by Annexin V binding by flowcytometry. A representative experiment of each transduction is shown(n=3 patients/transduction). (D) The percentage of HbFhigh cells wasassessed by flow cytometry on mature cells (GFP⁺ and low forward lightscatter-FSC). A representative experiment of each transduction is shown(n=3 patients/infection).

FIG. 5A and B. The kinetics of differentiation of β-TM and controlprogenitors. CD36 cells derived from CD34⁺β-TM peripheral blood cells(n=16 independent experiments, 7 patients) or healthy peripheral bloodcells (controls, n=8) were cultured with a two-phase amplificationliquid culture system, as described. Day 0 of differentiation is the 18start of CD36⁺ cell culture, which represents the differentiation phaseof the culture system. Differentiation staining was assessed daily byflow cytometry. (A) Upper panel: differentiation curves assessed byGlycophorin A (GpA) mean fluorescence intensity (MFI). Lower panel:differentiation curves assessed by c-KIT/CD 117 staining meanfluorescence intensity (MFI). The results are means (±SEM). p valuescompare β-TM and control-derived cells differentiated at day 8 and werecalculated using t-tests. *p<0.05, ***p<0.001. (B) One representativeflow cytometry image of the stage of erythroid differentiation at day 8of culture is shown for β-TM (upper panel) and control-derived cells(lower panel). GpA⁺/CD117⁻ cells represent mature erythroid cells.

FIG. 6. The lentiviral transduction of β-TM CD34⁺ cells with theβ-globin gene rescues Hsp70 nuclear localisation and GATA-1 protection.β-TM CD34+ cells derived from peripheral blood cells were infected witha lentivirus encoding the β-globin gene or a control virus. They werethen cultured with a two-phase amplification liquid culture system, asdescribed. Confocal microscopy analysis at day 8 of the CD36⁺ cellcultures shows Hsp70 nuclear localisation and GATA-1 expression inβ-globin gene transduced cells (top) and control transduced cells(bottom). Scale bar, 10 μm.

DETAILED DESCRIPTION

Methods and compositions for increasing erythrocyte maturation inindividuals suffering from β thalassemia are provided. The methodsinvolve preventing the otherwise untoward effects of Hsp70 sequestrationin the cytoplasm of maturing erythrocytes. The invention takes advantageof the new understanding of the mechanism behind the β-TM diseaseprocess as described herein, in order to provide methods andcompositions for increasing the maturation of erythrocytes containingHbF in β-TM subjects.

The inventors have found that symptoms of β-TM can be alleviated bycompositions and methods which slow or prevent (e.g. interfere with,impede, stop, decrease, etc.) cleavage and inactivation of GATA-1, orconversely, which preserve or promote the erythrocyte maturationactivity of GATA-1. Several avenues of doing so are provided.

In one aspect, GATA-1 inactivation is prevented by disrupting formationof the Hsp70/α globin complex in the cytoplasm, thereby increasing theconcentration of Hsp70 in the nucleus of β-TM cells, increasing nuclearlocalization of Hsp70, reducing maturation arrest and increasing thenumber of HbF cells. Using both wet and in silico chemistries, the Hsp70α globin binding site has been elucidated, and it has been surprisinglydiscovered that the binding site may be blocked or altered in ways thatprevent α globin binding but that do not impair the ability of Hsp70 tofulfill other functions in the cell, such as protecting the GATA-1protein from proteolysis.

Thus, in some aspects, the invention provides compositions and methodsfor blocking the α Hb binding site of Hsp70, without interferingsignificantly with the ability of Hsp70 to enter or access the nucleus,bind GATA-1 and protect it from proteolytic degradation, e.g. bycaspase-1. Blockage of the a Hb binding site may be carried out bycontacting Hsp70 in β-TM cells with a ligand that binds with relativelyhigh affinity to the α globin binding site, the affinity usually beingat least equal to or greater than that of α globin i.e. the Kd of theligand is approximately equal to or lower than that of α globin. Bindingof the ligand may be competitive so that α globin is outcompeted and theequilibrium distribution of Hsp70 between the cytoplasm and nucleus isshifted, and so that at least a portion of the Hsp70 that is present inthe cell is not complexed to α globin in the cytoplasm. Alternatively,ligand binding may be irreversible so that Hsp70 that binds the ligandcannot bind α globin, but can still access and bind GATA-1 protein inthe nucleus.

Ligands which may be used in the practice of the invention include butare not limited to various so-called “small molecules”. A “smallmolecule” is generally a low molecular weight (<900 Daltons organiccompound with a size on the order of 10⁻⁹ m. In general, the uppermolecular weight limit for a small molecule is approximately 900 Daltonswhich allows for the possibility to rapidly diffuse across cellmembranes and reach intracellular sites of action. In addition, thismolecular weight cutoff is a favorable (although insufficient) conditionfor oral bioavailability. In some aspects, lower molecular weightcompounds may be used, e.g. compounds of about 100, 200, 300, 400, 500,600, 700 or 800 Daltons or less. The compound may, but does not always,obey “Lipinski's rule”, which states that, in general, an orally activedrug has no more than one violation of the following criteria:

-   -   1. Not more than 5 hydrogen bond donors (nitrogen or oxygen        atoms with one or more hydrogen atoms)    -   2. Not more than 10 hydrogen bond acceptors (nitrogen or oxygen        atoms)    -   3. A molecular mass less than 500 daltons    -   4. An octanol-water partition coefficient [5] log P not greater        than 5. Such “small molecule” drugs (active agents) are        generally designed to interact with amino acid residues and/or        with functional groups thereof, located in the α chain binding        pocket of Hsp70. By the “α chain binding pocket” of Hsp70, we        mean the highly electronegative cavity formed by the N-terminal        nucleotide binding domain (NBD), the C-terminal substrate        binding-domain (SBD) and the lid (a C-terminal 10 kDa helical        subdomain of the SBD) of Hsp70. Suitable small molecules may be,        for example, small proteins or peptides, nucleic acids,        carbohydrates, antibodies, and suitable fragment thereof.        Alternatively, the small molecules may be a chemically        synthesized organic or inorganic molecule that is purposefully        designed to fit the α chain binding pocket of Hsp70.

In further embodiments, the invention provides methods identifyingcompounds or agents (e.g. small molecules) that inhibit binding of αglobin to Hsp70, e.g. methods of designing, generating and screeninggroups of agents in order to identify those which bind to, in, at ornear the α chain binding pocket of Hsp70 in a manner that prevents Hsp70from binding to α globin, or that lessens the affinity of Hsp70 for αglobin. Those of skill in the art are familiar with techniques forso-called rational drug design, e.g. designing, synthesizing, andscreening libraries of compounds that may have a desired activity, andthen analyzing results obtained in order to select suitable ligands foruse. Typically, information concerning the binding properties of themolecule to which the agent will bind is used to design, e.g. in silico,one or more suitable molecular “skeletons” or “frameworks” or“pharmacores” a possessing minimal properties required to bind to thetarget designed to “fit” a binding pocket, or a site adjacent to abinding pocket, or an allosteric site distant from the binding pocketbut which communicates structural changes across the molecule to thebinding pocket when an agent is bound thereto, etc. Various atoms oratomic groups are then added, in silico, to the basic framework in asystematic fashion, e.g. by first adding first a H atom, then a methylgroup, then an ethyl group, etc. to increase the length of a variablegroup at one or more positions by one CH₂ group at a time; or a batteryof positively or negatively charged groups may be placed at one or morepositions, etc. Once candidate compounds or families thereof aredesigned in silico, various computer implemented programs can be used toidentify the most likely candidates or families of molecular candidates,e.g. those which appear to possess statistically suitable bindingaffinities. In the present invention, such ligands must also appear tonot prevent the ability of Hsp70 to bind to and protect GATA-1 fromcaspase-1, or at least to still allow sufficient binding to Hsp70 toGATA-1 to provide a suitable positive outcome when administered to apatient by lessening disease symptoms.

Alternatively, or in addition to (e.g. before, after or during) theprocess of drug design, high throughput screening (HTS) data may be usedto rapidly identify active compounds of interest that bind to HSP-70.The results of HTS experiments may provide starting points for drugdesign, and/or confirmation of previous in silico drug design results,and/or may provide additional understanding of the interaction of HSP-70and the candidate ligands.

Once suitable candidate ligands or families of candidate ligands areidentified, synthesis of larger quantities of compounds of interest isaccomplished by methods known in the art, e.g. by a suitable chemicalsynthetic routes. Such molecules are then tested (e.g. in vitro, in vivousing animal models, and during clinical trials) by methods known tothose of skill in the art, e.g. further HTS using HSP-70 as the target,or further testing to elucidate specific attributes of the compounds(binding affinity, bioavailability, toxicity, stability, etc.).

The invention thus also provides method of screening candidate compoundsin order to select compounds which inhibit binding of α globin to Hsp70but which do not inhibit binding of Hsp70 to GATA-1. The methods maycomprise, for example steps such as: i) providing a plurality ofcandidate compounds which may inhibit binding of α globin to Hsp70; ii)exposing Hsp70 to said plurality of candidate compounds in the presenceof a globin and under conditions which allow α globin to bind to Hsp-70;iii) identifying Hsp70-compound complexes which do not contain bound αglobin; iv) exposing Hsp70-compound complexes identified in saididentifying step iii) to GATA-1 under conditions that permit Hsp70 tobind to GATA-1; and v) identifying Hsp70-compound-GATA-1 complexesformed in said exposing step iv); and vi) selecting compounds identifiedin said identifying step v) as compounds which inhibit binding of αglobin to Hsp 70 but which do not inhibit binding of Hsp70 to GATA-1.The method may also comprise a step of exposing Hsp70-compound-GATA-1complexes to caspase-1, and identifying Hsp70-compound-GATA-1 complexesin which GATA-1 is not proteolytically cleaved. In some aspects, thefirst exposing step may be carried out in two steps such as firstexposing HSP70 to candidate compounds and identifying complexes formedbetween HSP70 and compounds, and then exposing the HSP70-compoundcomplexes to α globin and selecting HSP70-compound complexes that do notbind α globin, or from which the compound is not displaced by α globin.Whatever the order of the screening steps, the compounds that areselected for clinical use (positive “hits”) must interfere with orprevent or inhibit or decrease or compete with the ability of HSP70 tobind α globin, and yet not interfere with or prevent or inhibit ordecrease or compete with the ability of HSP70 to bind toHsp70-compound-GATA-1, and also confer protection from proteolysis ofHsp70-compound- GATA-1 by caspase-1. Those of skill in the art arefamiliar with various statistical tools that can be used to assess thesignificance of such data, compared to that obtained with suitablecontrols.

Steps of the screening method (e.g. identifying, selecting) may becarried out by attaching to or incorporating into one or more of thesubstances being tested (e.g. HSP-70 and/or α globin and/or the compoundbeing tested and/or GATA-1 protein) a detectable label including but notlimited to: a radioactive moiety; a bioluminescent, chemiluminescent orfluorescent label; an affinity label or tag; etc. Further, one or moreof the substances may be immobilized on a substrate such as a plate orbead and the screening assays may include suitable steps of washing toremove unreacted substances, separation via size exclusion or affinitychromatography or by filtering, centrifugation, etc. Characterization ofidentified complexes of interest or confirmation of the identity may becarried out by known techniques, e.g. sequencing, reaction withantibodies, mass spec, etc.

The steps of the screening assays are carried out using suitableconcentrations of each reactant. Viable candidates for further testingand for clinical use will typically have binding affinities (Kd values)for HSP-70 in the range of at least about 25%, and usually 30, 35, 40,45, 50, 55, 60, 65, 70,. 75, 80, 85, 90, 95% or more of that of αglobin, compared to a suitable control, and the binding affinity mayequal or exceed that of α globin. When bound to HSP-70, a selectedcompound will typically reduce the binding of α globin to HSP-70 by atleast about 25%, and usually by 30, 35, 40, 45, 50, 55, 60, 65, 70,. 75,80, 85, 90, 95% or more, compared to a suitable control, and binding tomay be completely prevented. When bound to an HSP-70-compound complex,GATA-1 will typically be protected so as to reduce proteolysis by atleast 25%, and usually by 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95% or more, compared to a suitable control, and the binding ofα globin to HSP-70 may be completely prevented.

The present invention provides compositions for use in methods ofincreasing erythrocyte maturation in individuals or subjects in needthereof, particularly those with β-TM. The compositions include one ormore substantially purified active agents that promote erythrocytematuration as described herein, and a pharmacologically suitablecarrier. The preparation of such compositions for administration to amammal is well known to those of skill in the art. Typically, suchcompositions are prepared either as liquid solutions or suspensions,however solid forms such as tablets, pills, powders and the like arealso contemplated. Solid forms suitable for solution in, or suspensionin, liquids prior to administration may also be prepared. Thepreparation may also be emulsified. The active ingredients may be mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredients. Suitable excipients are, for example,water, saline, dextrose, glycerol, ethanol and the like, or combinationsthereof. In addition, the composition may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and the like. If it is desired to administer an oral form of thecomposition, various thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders and the like may be added. The composition ofthe present invention may contain any such additional ingredients so asto provide the composition in a form suitable for administration. Thefinal amount of active agent in the formulations may vary. However, ingeneral, the amount in the formulations will be from about 1-99%.

The active agent compositions (preparations) of the invention may beadministered by any of the many suitable means which are well known tothose of skill in the art, including but not limited to: by injection,(e.g. intravenous [IV], intraperitoneal, intramuscular, subcutaneous),by inhalation, orally, intravaginally, intranasally, by ingestion of afood or probiotic product containing the agent, etc. In preferredembodiments, the mode of administration is orally or by injection or IV.In addition, the compositions may be administered in conjunction withother treatment modalities such as various chemotherapeutic agents, ironsupplements, blood transfusions, agents that activate γ chain expression(e.g. that cause or promote transcription or translation of Hb γ chain),and the like.

For administration of genes encoding one or more (at least one) activeagent(s) as described herein, various options may be implemented. Insome asepcts, nucleic acids comprising sequences encoding an activeagent of the invention or functional derivatives thereof, areadministered to prevent, manage, treat and/or ameliorate β thalassemiaby way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acidwhich encodes the active agent. Generally, the encoding region isoperably linked to one or more expression control sequences, e.g.promoters, enhancers, etc. The sequence may be linked to other sequencessuch as STOP codons, and the like, in order to enable transcription ofthe gene into funcation mRNA, or, if RNA is administered, to enabletranslation thereof into an active form (or possibly a precursor of anactive form) of the active agent. The active agent, once fullyexpressed, mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow. For general review of the methods of gene therapy, see Goldspielet al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N.Y.(1990).

In some embodiments, a composition of the invention comprises nucleicacids encoding an active agent of the invention, said nucleic acidsbeing part of an expression vector that expresses the active agent inthe host to whom it is administered. In particular, such nucleic acidshave promoters, e.g. heterologous promoters, operably linked to thecoding region, the promoter being inducible or constitutive, and,optionally, tissue- or cell-specific. In other embodiments, nucleic acidmolecules are used in which the coding sequences and any other desiredsequences are flanked by regions that promote homologous recombinationat a desired site in the genome, thus providing for intrachromosomalexpression of the antibody encoding nucleic acids (Koller and Smithies,1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989,Nature 342:435-438).

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In some embodiments, the nucleic acid sequences are administered invivo, where the sequences are expressed to produce the encoded product.This can be accomplished by any of numerous methods known in the art,e.g., by constructing them as part of an appropriate nucleic acidexpression vector and administering the vector so that the sequencesbecome intracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cellsurface receptors or transfecting agents, encapsulation in liposomes,microparticles, or microcapsules, or by administering them in linkage toa peptide which is known to enter the nucleus, by administering it inlinkage to a ligand subject to receptor-mediated endocytosis (see, e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used totarget cell types specifically expressing the receptors), etc. In otherembodiments, nucleic acid-ligand complexes can be formed in which theligand comprises a fusogenic viral peptide to disrupt endosomes,allowing the nucleic acid to avoid lysosomal degradation. In yet anotherembodiment, the nucleic acid can be targeted in vivo for cell specificuptake and expression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180; WO 92/22635; WO 92/20316; WO93/14188, WO93/20221). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad.Sci. USA 86:8932-8935; and Zijlstra et al., 1989, Nature 342:435-438).

In some embodiments, viral vectors that contain nucleic acid sequencesencoding an active agent of the invention are used. For example, aretroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors contain the components necessaryfor the correct packaging of the viral genome and integration into thehost cell DNA. The nucleic acid sequences encoding the active agent tobe used in gene therapy can be cloned into one or more vectors, therebyfacilitating delivery of the gene into a subject. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdr 1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang et al., 1995, Gene Therapy 2:775-783. In apreferred embodiment, adenovirus vectors are used. Adeno-associatedvirus (AAV) has also been proposed for use in gene therapy (Walsh etal., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; and U.S. Pat. No.5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject. In the present invention, suchcells may be erythrocyles, e.g. immature erythroblasts. In thisembodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcellmediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (see, e.g., Loeffler and Behr, 1993, Meth. Enzymol.217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Clin.Pharma. Ther. 29:69-92 (1985)) and may be used in accordance with thepresent invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny. The resultingrecombinant cells can be delivered to a subject by various methods knownin the art. Recombinant blood cells (e.g., hematopoietic stem orprogenitor cells) are preferably administered intravenously. The amountof cells envisioned for use depends on the desired effect, patientstate, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc. In a preferred embodiment, the cell used for gene therapy isautologous to the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an active agent of the invention areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (see e.g., PCT Publication WO94/08598; Stemple and Anderson, 1992, Cell 7 1:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In some embodiments, the nucleic acid to be introduced for purposes ofgene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

In other embodiments, the active agent of the invention is not atranslatable peptide or protein but is e.g. an organic molecule that isdesigned and synthesized in a manner that confers on the molecule thefeatures that are necessary or desirable to promote its interaction withand binding to the negatively charged α chain binding pocket/cleft asdescribed herein. Methods of synthesizing molecules with e.g. chargedsurface atoms and/or surface atoms capable of hydrogen bonding areknown. For example, various condensation reactions are used to joinfunctional groups of interest, with or without the use of protectinggroups, and in the presence of suitable solvents, to generate linkedfunctional groups presenting desired atoms or groups of atoms in adesired location in the molecule. The molecules can be designed toinclude areas of rigidity and/or flexibility to accommodate the bindingpocket. Positioning and spacing of the atoms is such that the groups oratoms with which they are intended to interact in binding pocket will becontacted, or at least presented within bonding distance, whenintroduced into the binding pocket. Typically, at least 2, and usuallymore (e.g. 3, 4, 5, 6, 7, 8, 9, 10 or more, e.g. 15, 20, 25, 30 or more)specific binding interactions are planned, e.g. one positively chargedgroup to interact with one formal negative charge in the targetedbinding site, 1-3 hydrogen bond donors to interact with 1-3 otherhydrogen bond donors, etc. Suitable dimensions or ranges of dimensionsare generally determined in silico. Candidate active agents can bemanufactured and screened for activity using known methodology, e.g. viahigh throughput screening.

In some embodiments, the active agent is a small molecule that is apeptidomimetic. Peptidomimetic are small protein-like chains designed tomimic a peptide of interest. For the present invention, a peptide ofinterest is generally α globin or a portion thereof, e.g. the portion ofα globin that contacts and mediates binding to Hsp70. Peptidomimeticstypically arise either from modification of an existing peptide, or bydesigning similar systems that mimic peptides, such as peptoids andβ-peptides. Irrespective of the approach, the altered chemical structureis designed to advantageously adjust desirable molecular properties suchas stability, biological activity, ligand binding, etc. Themodifications generally involve changes to the peptide sequence that donot occur naturally, e.g. altered backbones, reduced peptide bonds,acylation of reactive groups, amidation of reactive groups,incorporation of nonnatural (non-proteinogenic or non-standard) aminoacids (e.g. D-amino acids, norleucine, lanthionine, 2-aminoisobutyricacid, dehydroalanine, gamma-aminobutyric acid, ornithine, citrulline,beta alanine (3-aminopropanoic acid), carnitine, hydroxyproline,selenomethionine, homocysteine, homoserine, and homophenyalanine,S-benzyl cysteine, etc. In addition, modifications such as sulfoniation,phosphorylation, etc. may be used to create the desired binding motif.

Herein, where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Before exemplary embodiments of the present invention are described ingreater detail, it is to be understood that this invention is notlimited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

EXAMPLES Example 1 Cytoplasmic Sequestration of Hsp70 by Excess of Freeα-Globin Promotes Ineffective Erythropoiesis in β-Thalassimia

It has been previously demonstrated that normal human erythroid cellmaturation requires a transient activation of caspase-36. AlthoughGATA-1, the master transcriptional factor of erythropoiesis, is acaspase-3 target, it has been shown that during human erythroiddifferentiation, it is protected from cleavage through its associationwith the chaperone heat shock protein 70 (Hsp70) in the nucleus⁷. Hsp70is constitutively highly expressed in normal human erythroid cells⁷. Thebest-known role of this ubiquitous chaperone is to participate inproteins folding and refolding of proteins denatured by cytoplasmicstress, thus preventing their aggregation⁸. Here, evidence is providedshowing that during the maturation of human β-TM erythroblasts, Hsp70 issequestrated in the cytoplasm by the excess of free α-globin chains,resulting in nuclear GATA-1 cleavage and, in turn, end-stage maturationarrest and apoptosis. A molecular modelling shows that α-globin binds toa highly electronegative cavity formed by all Hsp70 domains.Additionally, the transduction of a nuclear-targeted Hsp70 mutant(Hsp70-S400A) or caspase-3 uncleavable GATA-1 mutant (μGATA-1) correctsβ-thalassemia major IE in human cultured β-TM cells.

Methods

Erythroid cells were generated in vitro from the peripheral bloodcirculating CD34⁺ cells from 7 adult patients with β⁰-TM or 8 healthydonors. Fresh normal bone marrow cell smears were obtained from 3 adultpatients with β-TM who had undergone cholecystectomy or splenectomy, or3 healthy controls (allogenic bone marrow donors), after they had givenwritten informed consent. This study was performed according to theHelsinki Declaration with the approval from the ethics committee of ourinstitution. Erythroid cells in culture were generated as previouslydescribed^(6,7). Statistical analyses were performed using GraphPadPRIAM™ software. Data are expressed as the mean±standard deviation (SD)or median (interquartile range-IQR), unless noted otherwise. Student'spaired t-test or Mann-Whitney Utest was used as appropriate. *p value<0.05, **p value <0.01, ***p value <0.001

Erythroid Liquid Culture

The circulating peripheral blood of β-TM patients contains a smallnumber of hematopoietic progenitor cells¹⁹. Erythroid cells were alsogenerated in vitro from peripheral blood circulating CD34⁺ cells fromadult patients with β⁰ thalassemia major (β-TM), which were collectedbefore routine transfusion, or from control patients who were healthydonors treated with G-CSF to induce hematopoietic stem cellmobilisation. This study was done according to the Helsinki Declarationwith the approval from the ethics committee of the Comite de Protectiondes personnes (CPP) “Ile de France II”. All patients gave writteninformed consent. In the first step of culture (“cell expansion”),isolated CD34⁺ progenitors (Miltenyi CD34 Progenitor Cell Isolation Kit)were grown in the presence of 100 ng/ml IL-6, 10 ng/ml IL-3 and 100ng/ml SCF for 7 days. At day 7, CD36⁺erythroid progenitors were isolatedby magnetic isolation (Miltenyi Biotec). In the second phase of culture,which allows the “differentiation and maturation of erythroblasts”,CD36⁺ cells were cultured in the presence of 10 ng/ml IL-3, 100 ng/mlSCF and 2 U/ml Epo in IMDM (Gibco cell culture) supplemented with 15%BIT 9500 (Stem Cell Technologies), as previously described^(6,7).

Apoptosis Assay and Cell Differentiation

Apoptosis was assessed by Annexin V binding and propidium iodide (PI)staining (ebioscience). Early apoptotic cells were defined as Annexin-Vpositive and PI negative. Differentiation was assessed by variousmethods. First, morphological analysis after May-Grünwald-Giemsa (MGG)staining was used. Cells were examined under a Leica DMRB microscopewith a PLFluotar 40× oil objective in a blinded fashion. The number ofproerythroblasts, basophilic, polychromatic, acidophilic erythroblastsand reticulocytes was assessed in each experiment by countingapproximately 300 cells in consecutive oil immersion fields andexpressed as a percentage of total cells. Additionally, differentiationwas assessed by calculating a terminal maturation index on MGG, definedby the number of acidophils+reticulocytes per slide×100/number ofpolychromatophilic cells per slide. This allowed better characterizationof maturation arrest at the polychromatophilic stage, which is known tobe a hallmark of ineffective erythropoiesis⁴, and its modulation.Haemoglobin content was also measured, as assessed by benzidinestaining. Flow cytometry analysis was also performed each day afterdouble labelling with a phycoerythrin (PE)-conjugated anti-GPA (BDPharmingen) antibody and an APC-conjugated anti-CD117 (c-kit)(e-bioscience) antibody. The percentage of GPA+/CD117-cells representedmature erythroid cells20. F cells were evidenced by flow cytometryanalysis. The cultured cells were fixed and permeabilised, washed with1× PBS/1% BSA and then stained with PE-conjugated anti-human haemoglobinF (HbF) (BD Pharmingen) for 30 min at room temperature. The cells werethen analysed by fluorescence-activated cell sorting (FACS).

Analysis of the Hsp70/Alpha-Globin Complex Yeast Two-Hybrid Assay

The bait vector used was pGBKT7, and the prey vector was pGADT7(Clontech). The human α- and β-globin coding sequences were cloned intothe EcoRI/BamHI and NdeI/ClaI sites, respectively, of the pGADT7. Thecoding sequence of the human Heat Shock Protein 70 (Hsp70) was clonedinto the EcoRI/BamHI sites of the pGBKT7. An N-terminal nucleotidebinding domain (NBD) (residues 1-380) and a C-terminal substratebinding-domain (SBD) (residues 394-615) were cloned in pGBKT7. ThepGBKT7-p53 plasmid was used as a positive bait control. This plasmidencodes the GAL4 DNA-BD fused with murine p53. As a positive controlprey plasmid, pGADT7-SV40, which encodes the GAL4 activation domainfused with SV4021 large T-antigen, was used. The empty vector, pGADT7,was used as a negative control prey plasmid. All pGADT7- andpGBKT7-derived vectors were transformed into Y187 and Y2HGold yeaststrains, respectively (Clontech Yeastmaker Yeast Transformation System2). After growth at 30° C. for 3 to 5 days, the transformants wereselected on Leu_(—) and Trp_(—) minimal media plates, respectively. Eachprey strain was mated with the bait strain to generate diploid yeastcells (Clontech Matchmaker Gold Yeast 2-Hybrid system). Diploid yeastcells selected on Leu_Trp_(—) minimal media plates were then patchedonto Leu_Trp_(—) minimal media plates with X-α-Galactosidase (40 μg/mL)and Aureobasidin A (70 ng/mL). Blue diploid cells appeared after 3 to 5days at 30° C., indicating the interaction between the bait and the preyproteins. To confirm these results, the diploid yeast cells were thenpatched onto higher stringency His_Ade_Leu_Trp_(—) minimal media platessupplemented with 40 μg/mL X-α-Galactosidase and 70 ng/mL AureobasidinA.

Confocal Analysis Cell Permeabilisation and Labelling for FluorescenceMicroscopy.

The cells were washed, spun onto slides, fixed with acetone, hydratedwith cold 1× PBS/1% BSA for 30 minutes, treated with formaldehyde(Sigma) for 15 minutes, and then with methanol (Prolabo) for 10 minutesat room temperature. Next, the cells were permeabilised with 1× PBS/0.2%Triton X100 (Sigma) for 10 minutes at 4° C., washed with 1× PBS/1% BSAand incubated in 3% BSA for 30 min. They were then sequentiallyincubated with the antibodies as follows: anti-GATA-1 overnight at 4°C., anti-rat-Cy3 for 45 minutes at room temperature, rabbit anti-Hsp70or anti-caspase-3 for 1 hour at room temperature, anti-rabbit Alexa 647for 45 minutes at room temperature and anti-haemoglobin-FITC (Abcam) for60 minutes at room temperature. All antibodies were diluted in 1× PBS/1%BSA/0.1% Tween (Sigma). Nuclei were stained with DAPI, and the slideswere examined with a confocal laser microscope (LSM 510 Carl Zeiss).Fresh, normal, bone-marrow cell smears were fixed with acetone.Permeabilisation and labeling with anti-GATA-1, anti-Hsp70 and anti-αglobin antibodies were performed as above. To more precisely analyse theHsp70/α-globin interaction, the Duolink® II technology (Olink®Bioscience), which is an in situ proximity ligation assay technology,was used. In this assay, a pair of secondary antibodies labelled witholigonucleotides (PLA probes) only generates a signal when the twoprobes are bound in close proximity (<40 nm). The signal from eachdetected pair of PLA probes is visualised as an individual fluorescentspot²¹ . Slides were incubated with primary antibodies as describedabove and with secondary antibodies conjugated with oligonucleotides(PLA probe MINUS anti-mouse and PLA probe PLUS anti-rabbit). Ligationand amplification reactions were performed according to themanufacturer's instructions.

Molecular Modelling, Docking and Molecular Dynamics (MD) Simulations

All calculations were carried out on a PC station and on the Meso Centerof ENS de Cachan running Linux CentOs 5.2. Figures were produced withthe PyMOL molecular graphics system22, and R was used for statisticalcomputing²³. The template structures for homology modelling wereretrieved from the Protein Data Bank (PDB)24.

Molecular Modelling

-   -   Hsp70: The 3D model of the full-length human Hsp70¹⁻⁷⁰¹was        generated from partial structures (X-ray or RIVIN) by homology        modelling of the separate Hsp70 domains and molecular complex        Hsp110·Hsp70 (PDB codes: 3C7N 25, 2QWL 26, 3FE1 27 and 2LMG 28).        The sequence of Hsp70¹⁻⁷⁰¹was aligned to the template sequences        with ClustalW29 and Modeller30. The interdomain linkers were        constructed by ab-initio loop generation using Modeler.²³    -   α-globin: The initial structural data of α-globin was taken from        the X-ray structure (PDB code: 3S66 31) and was docked onto the        generated model of Hsp70¹⁻⁷⁰¹ using three algorithms: Zdock32,        FlexDock33 and HingeProt34. The minimised structure (CHARMM)35        of the Hsp70¹⁻⁷⁰¹/α-globin complex was analysed for hydrophobic        and electrostatic complementarities and Hbond interactions on        the interface of the proteins. The ADP-Mg²⁺ and Haem Fe²⁺ were        incorporated into proteins.

Molecular Dynamics (MD) Simulations

Molecular Dynamics (MD) simulations were performed using GROMACS 4.5.436 with CHARMM 27 force field. Each generated model, Hsp70 or theHsp70¹⁻⁷⁰/α-globin complex, was solvated in a TIP3P water box with aminimum distance of 15 Å from the edge of the box to any protein atom.The charges of the system were neutralised by adding counterions (Na⁺ orCl⁻). The solvated systems were first minimised for 1,000 steps with theprotein atoms restrained, followed by another 3,000 steps ofminimisation with all atoms allowed to move. The temperature of eachsystem was then increased to 300 K by increments of 0.001 K during 2 ps.The system was further equilibrated under constant volume andtemperature (NRT) conditions for 100 ps constraining protein backboneatoms, followed by 500 ps equilibration without constraints underconstant pressure and temperature (NPT) at 300 K and 1 bar. Productionsimulations were performed for 20 ns in the NPT ensemble. Short-rangeinteractions employed a switch function with a 12 Å cut-off and a 10 Åswitch distance, and the long-range electrostatic interactions werecalculated with the Particle Mesh Ewald protoco137. During productionsimulations, the time step was 2 fs, with a SHAKE constraint on allbonds containing hydrogen atoms.

Viral Transduction

Lentiviral Production

The nucleus-targeted Hsp70 mutant (Hsp70-S400A) was cloned in thepTrip_U3EF1 lentiviral vector upstream of an IRESECMV—Green FluorescentProtein (GFP) cassette. Infectious vector particles were produced in293T cells by cotransfection of the vector with the encapsidationplasmid psPAX2 and the expression plasmid pHCMV-G, using the jetPRIME™transfection reagent (Polyplus). Supernatants were collected 48 hoursand 72 hours after transfection and were pooled and concentrated byultracentrifugation. Virus stocks were kept frozen at −80° C. For thelentivirus production of the β-globin gene, vesicular stomatitis virusglycoprotein pseudotyped lentiviral supernatant was produced bytransient transfection of HEK293T cells with the 5-plasmid system(LentiGlobin construct, HPV 275-gag-pol plasmid, TN 15-vsvG env plasmid,p633-rev plasmid, HPV601-tat plasmid) by calcium phosphatecoprecipitation in Dulbecco's modified Eagle's media supplemented with5% foetal bovine serum (Invitrogen), followed by harvest in CellGro SCGMserum-free media (CellGenix) after 48 h. Concentrated virus was thenfrozen and stored at −80° C.

Retroviral Production

Uncleavable mutant GATA-1 (μGATA-1) was cloned in PINCO vector upstreamof a CMV promoter-Green Fluorescent Protein (GFP) cassette. These wereused to produce vector particles by cotransfection of 293EBNA cells withthe vector plasmid, an encapsidation plasmid (gag-pol) lacking allaccessory HIV-1 proteins, and an expression plasmid (pHCMV-G) encodingthe vesicular stomatitis virus (VSVg) envelope, using jetPRIME™transfection reagent.

Infection of Erythroid Cells

Hsp70-S400A and μGATA-1 transduction: CD34+cells isolated from β-TMperipheral blood mononuclear cells were cultured for 5 days, asdescribed above. They were then infected by lentiviruses orretroviruses, in the presence of 4μg/ml protamine sulphate. A secondround of infection was performed 24 hours later, upon changing to freshmedium with cytokines. After an additional 24 hours, cells wereextensively washed in PBS and stained with the anti-CD36-APC mAb (BDPharmingen). The CD36⁺/GFP⁺ cell population was purified by cell sortingand cultured for 7 to 10 additional days in serum-free medium in thepresence of IL3⁺SCF⁺EPO, as described above.

β-Globin Gene Lentivirus Transduction

CD34⁺ cells were isolated from β-TM peripheral blood mononuclear cellsby magnetic sorting (Miltenyi Biotec). Sorted cells were prestimulatedfor 24 h in CellGro SCGM media supplemented with 100 ng/mL hSCF, 100ng/mL hTPO, 100 ng/mL hFIt3L and 60 ng/mL hIL-3 at 37° C. and 5% CO2.Then, prestimulated cells were transduced for 22 h with the LentiGlobinvector at an MOI of 50 in CellGro SCGM media supplemented with 100 ng/mLhSCF, 100 ng/mL hTPO, 100 ng/mL hFlt3L, 60 ng/mL hIL-3 and 4 μg/mLprotamine sulphate, or mock transduced in the same conditions. Two-phaseliquid culture was then performed as described above.

Statistical Analyses

Statistical analyses were performed with GraphPad Prism™ (version 5.0;GraphPad Software). The data are expressed as the mean standarddeviation (SD) or median (interquartile range-IQR), unless notedotherwise. Student's paired t-test or Mann-Whitney U-test was used asappropriate. *p value <0.05, **p value <0.01, ***p value <0.001

RESULTS

To investigate the hypothesis that Hsp70 can be sequestrated in thecytoplasm of mature β-TM erythroblasts by binding to free α-globinchains of haemoglobin or aggregates, its subcellular localisation wasanalyzed in fresh bone marrow samples from adult TM patients (n=3) andhealthy donors (n=3) by confocal microscopy. Erythroid maturation wasevaluated by cell size and by α-globin staining intensity. The resultsshowed that Hsp70 was mainly localised in the cytoplasm and that GATA-1was poorly expressed in the nucleus of mature haemoglobinisederythroblasts from β-TM patients, in contrast to controls (FIG. 1A).

Next, to decipher the role of Hsp70 in ineffective erythropoiesis inβ-TM, an in vitro two-phase amplification liquid culture was performed,allowing the proliferation, survival and erythroid differentiation ofβ-TM (n=16) or control CD34+(n=8) progenitors towards the formation ofacidophilic erythroblasts and reticulocytes. During the first phase ofamplification, cell proliferation did not differ between thalassemic andcontrol cells. In contrast, during the second phase, corresponding toerythroid terminal differentiation and maturation, at day 8, weobserved, in β-TM, an accelerated differentiation characterised by ahigher percentage of polychromatophilic cells (26.2%*8.4 vs. 9.0%±2.7;p=0.003) (FIG. 2A), an accelerated down regulation of the earlyerythroid marker KIT/CD117 (mean fluorescence intensity (MFI) 25.7±17vs. 115.1±47.1; p=0.0001) and an up regulation of GPA (MFI 243.8±87.8vs. 178.9±56, p=0.04) (FIGS. 5A and B). At the time of intensehaemoglobinisation (d8-d10), in β-TM cells, apoptosis was increased (atd10, 38.2%±15.1 vs 18.5%±8.7; p=0.01) (FIG. 2B) and terminal maturationwas arrested at the polychromatophilic stage. To quantify thismaturation arrest, an index of terminal maturation was defined as thenumber of (acidophilic cells+reticulocytes per slide)×100/number ofpolychromatophilic cells per slide. At day 8, this index wassignificantly decreased in β-TM cells, to 16% (IQR 8.8-27.8) compared to37.6% in control cells (IQR 24.4-70.7; p=0.009) (FIG. 2A). Takentogether, this system of cell culture reproduced the characteristics ofIE observed in β-TM, namely accelerated differentiation, maturationarrest and the death of mature haemoglobinised cells³⁻⁵. Next, thesubcellular localisation of Hsp70 was analysed at several time intervalsin β-TM patients (n=7) and healthy donors (n=7) by confocal microscopy.In agreement with what we observed in fresh primary bone marrowerythroblasts, Hsp70 was detected in the nucleus of control cells butwas absent or only weakly expressed in mature haemoglobinised β-TMcells. Thus, at day 8, the ratio of cytosoplasmic/nuclear Hsp70 MFI inβ-TM erythroblast cells was significantly increased, with a median ratioof 2.3 (IQR 1.6-3) compared to 1.1 in control cells (IQR 0.7-1.6;p<0.0001) (FIG. 2C). As a result, GATA-1 was poorly expressed in thenucleus of mature haemoglobinised β-TM erythroblasts (FIG. 2C), thussupporting the hypothesis. To further analyse and understand the linkbetween haemoglobinisation, Hsp70 localisation and the decrease inGATA-1 expression, the changes in the expression of these proteinsduring differentiation and maturation was studied. In β-TM derivedcells, the intensity of nuclear Hsp70 and GATA-1 staining decreased witherythroid differentiation and maturation, while these increased incontrols (FIG. 2D).

To demonstrate that Hsp70 could act as a molecular chaperone of freeα-globin chains, the subcellular localisation of both proteins was firstanalyzed by co-immunofluorescence experiments (n=7). From day 6 ofculture, Hsp70 and α-globin were co-localised in the cytoplam of β-TMerythroblasts. Features of aggregates were sometimes observed indifferentiated, haemoglobinised cells, with one typical picture shown inFIG. 2C. Co-localisation was assessed using the average Pearson'scorrelation coefficient (PCC). At day 8, an apparent high level ofco-localisation between Hsp70 and α-globin was detected, both in β-TM(PCC=0.4±0.13) and in controls (PCC=0.31±0.09). This finding wasconfirmed by Van Steensel's approach (data not shown). Similar findingswere observed in fresh bone marrow experiments from patients and healthydonors (data not shown). To further characterise this co-localisation, aclose in situ proximity ligation assay we used (Duolink®), which allowsthe identification of interacting proteins by fluorescent spots. At day8, spots we detected in β-TM mature haemoglobinised cells (n=3) but muchless so in controls. Additionally, cells containing abundantHsp70/α-globin complexes were apoptotic (FIG. 3A).

Next, using a yeast two-hybrid system, additional evidence for thebiochemical interaction of Hsp70 with the human α-globin chains wasprovided. In the assays, the entire coding sequence of human Hsp70 wasused as bait. Blue diploid transformants could be detected on a highstringency minimal medium, indicating a direct interaction between Hsp70and the α-globin chains Similar results were obtained when the β-globincoding sequence was used as prey, indicating that both the α- andβ-globin chains could interact with Hsp70 (FIG. 3B). To identify theHsp70 domains involved in this interaction, we tested the binding ofα-globin chains to deletion mutants of Hsp70 that expressed either theNucleotide Binding Domain (NBD) or the Substrate Binding Domain (SBD) ofHsp70 (FIG. 3B). Interestingly, neither of these two deletion mutantsinteracted with the α-globin chain, suggesting that the entire structureof Hsp70 is required for the recognition of α-globin. To bettercharacterise this interaction, an in silico study involving molecularmodelling, docking and molecular dynamics simulations was performed. Theα-globin was docked onto a homology-generated model of Hsp70¹⁻⁷⁰. It wasfound that α-globin binds to a highly electronegative cavity formed bythe NBD, SBD and lid (a C-terminal 10 kDa helical subdomain of the SBD)(FIG. 3C). The resulting Hsp70¹⁻⁷⁰/α-globin complex is stabilised byextensive protein-protein interactions mediated mainly by multiplehydrogen bonds engaging the three structural domains of Hsp70 (data notshown). Thus, the binding of α-globin to Hsp70 crucially modulates thechaperone structure and the interdomains interaction between NBD-lid andNBD-SBD. Altogether, these findings indicate that, in addition to AlphaHaemoglobin Stabilising Protein (AHSP), which stabilises the a -globinchains⁹, Hsp70 could act as a novel chaperone of α-globin chains.However, this apparent cytoprotective function of Hsp70 might bedetrimental during stages of high haemoglobinisation and globin chainimbalance in β-TM by preventing the nuclear localisation of Hsp70 and,consequently, its function in protecting GATA-1 from cleavage bycaspase-3.

To investigate the contribution of Hsp70 cytoplasmic sequestration tothe pathophysiology of β-TM IE, lentiviral transduction we used torestore Hsp70 expression in the nucleus of β-TM erythroblasts. For thispurpose, β-TM CD34+ cells (n=3) were transduced with lentivirusesexpressing a nuclear-targeted Hsp70 mutant (Hsp70-S400A) 10 wtHsp70, oran empty lentivector. As expected, at day 7 of the differentiation phaseof culture, Hsp70-S400A (FIG. 4A) and wtHsp70 (not shown) lentivectorsincreased nuclear Hsp70 localisation and rescued GATA-1 expressioninn-TM erythroid cells. The restoration of nuclear Hsp70 localisationefficiently improved the terminal maturation of β-TM erythroblasts. Atday 7, in Hsp70-S400A transduced β-TM erythroblasts, the percentage ofmature cells (acidophilic cells and reticulocytes) was increased whencompared to the empty vector control (10.6±3.2% vs 1.1±0.7%, p=0.01 FIG.4B). Similarly, the terminal maturation index was increased (28.1% (IQR28.1-51.3) vs 4.6% (IQR 1.7-6.7); p=0.01). In addition, rescuing nuclearHsp70 localisation induced a dramatic two-fold decrease in apoptosis(9.9±2.8% vs 20.7±5.6%; p<0.001) (FIG. 4C). Next, to analyse theconsequences of GATA-1 cleavage on the maturation arrest and apoptosisobserved in cultured β-TM erythroblasts, we transduced β-TM CD34+ cellswith a GATA-1 mutant that was uncleavable by caspase-3 (μGATA-1)¹¹ or aGFP+ empty vector. The μGATA-1 mutant had a positive effect on erythroidterminal maturation that was similar to that of Hsp70-S400A (FIG. 4A-D).Conversely, apoptosis was not corrected indicating that cleavage ofGATA-1 contributes to impair the erythroid maturation but to a lessextent to apoptosis of β-TM cells, as previously reported in low grademyelodysplastic syndromes¹⁰.

Foetal haemoglobin (HbF, cop), which is replaced after birth by theadult haemoglobin (HbA, α₂β₂), is concentrated in a few F cells andrepresents less than 1% of the haemoglobin content in healthy adults¹².In β-TM patients, there is an elevation in the proportion of F cells tocompensate for the lack of β-chain synthesis, and the only survivingmature erythroblasts are F cells. GATA-1 has a major role in regulatingthe haemoglobin gene expression; it is sometimes described as atranscription repressor or activator of the human γ-globin chainsgene¹⁴⁻¹⁶. As such, the effect of GATA-1 nuclear restoration on HbFexpression was studied by flow cytometry. At day 7, it was observed thatwhile the number of F cells decreased with maturation (data not shownand ¹⁷), the percentage of HbF^(high) cells, as assessed by flowcytometry, was significantly increased concomitantly with the protectionof GATA-1 by Hsp70-S400A (54.8%±12 vs 45.9%±10.5; p<0.004) (n=3) and inGATA-1 transduced erythroblasts (51.4±8.2% vs 40.5±9.4%; p<0.002) (n=3)(FIG. 4D). Finally, to ensure the specificity of these findings, β-TMCD34⁺ cells were lentivirally transduced with the β-globin gene. Thisled to Hsp70 nuclear re-localisation, GATA-1 protection (FIG. 6), andthe restoration of normal erythroid maturation (data not shown).

Taken together, our data demonstrate that the cytoplasmic sequestrationof α-globin chains by Hsp70 prevents their nuclear localisation. This isa key mechanism inducing the IE observed in β-TM patients. The modellingstudies suggest that Hsp70 could have been selected during evolution toserve as a specific chaperone of globin chains to protect earlyerythroblasts during erythroid differentiation.

The structural model of the Hsp70¹⁻⁷⁰/α-globin complex provides a newrationale for a targeted therapy in β-thalassemia major IE. Smallcompounds disrupting the Hsp70/α-globin complex in the cytoplasm mayincrease the nuclear localisation of Hsp70 and may thus reducematuration arrest and increase the number of F cells. Ultimately, theseoutcomes may decrease the patients' requirement for a blood transfusionand the associated complications, including iron overload.

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While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1. A method of restoring or increasing erythrocyte maturation in asubject suffering from β-thalassemia major (β-TM) by preventingproteolytic inactivation of GATA-1.
 2. The method of claim 1, whereinsaid preventing is achieved by administering to said subject a compoundthat inhibits binding of α globin to Hsp70.
 3. The method of claim 2,wherein said compound that inhibits binding of α globin to Hsp70 is asmall molecule that binds to the α chain binding pocket of Hsp70. 4-5.(canceled)